Expression of Exogenous Proteins in Donor Platelets Treated with Lipid Nanoparticles

This patent application claims the benefit of priority from U.S. Provisional Patent Application 63/344,247 filed May 20, 2022; the entire contents of which are incorporated herein by reference.

This invention was made with Government support under W81XWH-20-2-0041 awarded by the Medical Research and Development Command. The government has certain rights in the invention.

The present invention discloses novel lipid nanoparticle compositions useful for the transfection and expression of exogenous proteins in platelet cells.

Platelets are an integral component of hemostasis and donor platelets are routinely transfused to restore hemostatic balance in thrombocytopeniar actively bleeding patients. Platelets also carry potential as a cell therapy-based treatment for non-coagulopathic diseases; for example, they directly interact with cancer cells to promote tumour growth and metastasis, an association that can been exploited to develop platelet-based targeted cancer therapeutics. Platelet-derived extracellular vesicles have also been proposed as drug delivery vehicles due to specific organ targeting. Despite crucial roles in both hemostasis and cell-specific interactions, and thus significant potential as a target cellular therapy, there is a paucity of technologies capable of enhancing platelet biochemistry through genetic modification.

Despite being anucleate, platelets actively synthesize proteins both in vivo and once isolated from donor blood, making them an attractive candidate cell for genetic engineering.This pathway presents an attractive target for genetic engineering, to tune platelet function through protein expression. Existing methods of engineering platelets are indirect and involve transfection of hematopoietic stem cell platelet precursors to produce progenitor platelets with modified function. Two approaches include (1) ex vivo production of platelets from stem cells, which is limited by scalability, and (2) in vivo transfection of bone marrow megakaryocytes which is limited by transfection agent accessibility and specificity.Direct approaches of modifying donor platelet functions are even more limited. Viral based transfection strategies are not useful for direct donor platelet transfection since platelets lack a nucleus and the ability to synthesize their own mRNA transcripts from DNA. Physical and chemical transfection approaches may effectively deliver nucleic acid into platelets; however, protein expression remains to be conclusively observed. Chemical transfection methods such as lipofectamine fails to induce sufficient mRNA uptake by platelets and translation is not observed.Electroporation, a physical transfection approach that induces small pores in the cell plasma membrane to enable cargo uptake, has not reliably demonstrated protein expression from delivered mRNA.

Lipid nanoparticles (LNP) are a flexible and clinically approved platform which enable delivery and intracellular release of genetic material. Despite being anucleate, and thus unamenable to DNA-based transfection technologies, platelets contain all the translational machinery necessary for protein synthesis. Platelets are therefore ideal for modification by mRNA-containing LNP (mRNA-LNP) to enable expression of exogenous protein.

Advancements in lipid nanoparticle (LNP) technology have dramatically improved the delivery efficiency of mRNA into target cells. LNPs are currently used in the FDA approved siRNA therapeutic, Onpattro, and the Pfizer and Moderna COVID-19 vaccines.Genetically engineering donor platelets using lipid nanoparticles and RNA to produce exogenous protein ex vivo will enable the deliberate modulation of platelet function, or the introduction of new functions that will expand platelets as a cell therapy for therapeutic purposes.

The present invention relates to a lipid nanoparticle (LNP) composition for expression of exogenous proteins in a population of transfected platelet cells.

In a first aspect, the invention relates to a lipid nanoparticle (LNP) composition for expression of an exogenous protein in a population of transfected platelet cells, wherein the LNP composition comprises mRNA encoding the exogenous protein and a lipid mixture, and the lipid mixture comprises at least one ionizable cationic lipid, at least one helper lipid, a sterol, and at least one polyethylene glycol (PEG)-lipid conjugate.

In some embodiments, the at least one ionizable cationic lipid in the lipid mixture is CL4H6, SM-102, ALC-0315, CL1H6, CL15H6, CL1D6, ALC-0159, or any combination thereof. In other embodiments, the at least one ionizable cationic lipid is CL4H6 or ALC-0315.

In some embodiments, the at least one helper lipid in the lipid mixture is phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM), or any combination thereof. In other embodiments, the at least one helper lipid in the lipid mixture is phosphatidylcholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

In some embodiments, the at least one ionizable cationic lipid in the lipid mixture is CL4H6 and the helper lipid is phosphatidylcholine (POPC). In other embodiments, the at least one ionizable cationic lipid in the lipid mixture is ALC-0315 and the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

In some embodiments, the sterol is cholesterol or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, beta-sitosterol, fucosterol, or any mixture thereof. In other embodiments, the sterol is cholesterol.

In some embodiments, the at least one polyethylene glycol (PEG)-lipid conjugate is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE-PEG2000), or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG). In other embodiments, the at least one polyethylene glycol (PEG)-lipid conjugate is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

In another aspect, the invention relates to a lipid nanoparticle (LNP) composition for expression of an exogenous protein in a population of transfected platelet cells, wherein the LNP composition comprises messenger RNA (mRNA) encoding the exogenous protein and a lipid mixture, wherein the lipid mixture comprises 30-55 mole percent of the at least one ionizable cationic lipid, 5-20 mole percent of the at least one helper lipid, 25-50 mole percent of a sterol; and 0.5-3 mole percent of the at least one polyethylene glycol (PEG)-lipid conjugate.

In one embodiment, the lipid mixture comprises 50 mole percent of the at least one ionizable cationic lipid, 10 mole percent of the at least one helper lipid, 38.5 mole percent of a sterol; and 1.5 mole percent of the at least one polyethylene glycol (PEG)-lipid conjugate. For example, the lipid mixture may comprise 50 mole percent CL4H6 or ALC-0315 ionizable cationic lipid, 10 mole percent of phosphatidylcholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) helper lipid, 38.5 mole percent of cholesterol; and 1.5 mole percent of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

In yet another aspect, the messenger RNA present in the LNP composition may comprise one or more nucleotide modifications. In some embodiments, the modified nucleotide is 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine.

In some embodiments, the mRNA in the LNP composition may encode an exogenous protein such as: a coagulation factor, an anti-fibrinolytic protein, an anticoagulant, a fibrinolytic protein, an antimicrobial protein, an immunomodulator, an anti-cancer protein, a genetic editing protein, or a reporter protein. In other embodiments, the mRNA in the LNP composition is a non-coding RNA or DNA aptamer.

In another aspect, the present invention relates to a lipid nanoparticle (LNP) composition for expression of an exogenous protein in a population of transfected platelet cells, wherein the LNP composition comprises mRNA encoding the exogenous protein and a lipid mixture, the lipid mixture comprising at least one ionizable cationic lipid, at least one helper lipid, a sterol, and at least one polyethylene glycol (PEG)-lipid conjugate, wherein the mRNA encoding the exogenous protein may comprise one or more modified nucleotides. For example, the mRNA encoding the exogenous protein may comprise 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine.

In still another aspect, the present invention relates to the use of the above LNP compositions for transfection of platelets with messenger RNA encoding an exogenous protein and expression of the exogenous protein in the resulting transfected platelets.

In some embodiments, the present invention relates to use of an LNP composition wherein the ionizable cationic lipid in the lipid mixture is CL4H6 and the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). When such LNP composition is used to produce a population of transfected platelet cells expressing an exogenous protein, such population of transfected platelet cells exhibits only minimal activation, as evidenced by cell surface exposure of platelet activation marker CD62P at levels less than those seen in platelets prepared in parallel with transfected platelets, but receiving 0.1 U mLthrombin in lieu of LNP.

In a final aspect, the present invention relates to a population of platelets expressing an exogenous protein, wherein said population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the lipid mixture comprises at least one ionizable cationic lipid, at least one helper lipid, one or more sterol; and at least one polyethylene glycol (PEG)-lipid conjugate.

In some embodiments, the population of platelets is transfected with an LNP composition comprising a mRNA encoding an exogenous protein and a lipid mixture, wherein the at least one ionizable cationic lipid in the lipid mixture is CL4H6, SM-102, ALC-0315, CL1H6, CL15H6, CL1D6, ALC-0159, or any combination thereof. For example, the one ionizable cationic lipid is CL4H6 or ALC-0315.

In some embodiments, the population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the at least one helper lipid in the lipid mixture phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM), or any combination thereof. In other embodiments, the population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the at least one helper lipid in the lipid mixture is phosphatidylcholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

In some embodiments, the population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the sterol in the lipid mixture is cholesterol or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, beta-sitosterol, fucosterol, or any mixture thereof. In other embodiments, the population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the sterol in the lipid mixture is cholesterol.

In some embodiments, the population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the at least one polyethylene glycol (PEG)-lipid conjugate in the lipid mixture is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino (polyethylene glycol)-2000 (DSPE-PEG2000), or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG). In other embodiments, population of platelets is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the at least one polyethylene glycol (PEG)-lipid conjugate in the lipid mixture is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

In some embodiments, a population of platelet cells is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the at least one ionizable cationic lipid in the lipid mixture is CL4H6 and the helper lipid in the lipid mixture is phosphatidylcholine (POPC). In other embodiments, the population of platelet cells is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the at least one ionizable cationic lipid in the lipid mixture is ALC-0315 and the helper lipid is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

In some embodiments, the population of platelet cells is transfected with an LNP composition comprising a mRNA encoding the exogenous protein and a lipid mixture, wherein the messenger RNA present in the LNP composition comprises one or more nucleotide modifications, such as 5-methoxyuridine, pseudouridine, or N-methylpseudouridine.

The present invention discloses novel lipid nanoparticle compositions that are internalized by platelets, ex vivo, with supported intracellular mRNA release once inside, and downstream expression of the encoded protein.

“Activation” or “activated”, as used herein in the context of platelets, refers to ability of platelets to aggregate and promote blood clotting, as measured by any method or assay known to one of skill in the art. For example, activation of platelets may be measured by monitoring levels of cell surface exposure of CD62P, a marker of platelet activation, by flow cytometry.

“Cationic lipid”, as used herein, a protonatable tertiary amine (e.g., pH titratable) head group, C16 to C18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds.

“Ionizable cationic lipid” or “ICL”, as used herein, is a cationic lipid that is protonated and positively charged at low pH but remains neutral at physiological pH.

“Exogenous”, as used herein, refers to a messenger RNA (mRNA) or protein that is not naturally found in, or produced by, platelet cells.

“Helper lipid”, as used herein, refers to any vesicle-forming lipid (e.g., bilayer-forming lipid) lipid, other than the ICL, that is added to a lipid nanoparticle to enable or enhance the ability of the mRNA within the LNP to transfect a platelet cell.

“Lipid nanoparticle(s)” or “LNP(s)”, as used herein, are spherical vesicles made of one or more ionizable cationic lipid, one or more helper lipid, cholesterol (to fill the gaps between the lipids), and one more polyethylene glycol (PEG)-lipid conjugate, plus the nucleic acid (DNA or RNA) cargo within the particle.

“Minimal activation of platelets” or “minimally activated”, as used herein, refers to maintaining a state of platelet activation which is less than the level seen in platelets treated with agonist thrombin, following preparation for transfection.

“Mole percent” or “mol %”, as used herein, is the percentage of the moles of a particular component relative to total moles of all components that are in a mixture.

“Polyethylene glycol-lipid conjugate” or “PEG-lipid” or “PEGylated lipids”, as used herein, refers to derivatives of polyethylene glycol (PEG) covalently attached to a lipid moiety;

The present invention relates to novel LNP compositions useful for transfection of platelet cells with exogenous messenger RNA (mRNA-LNP) and expression of the encoded endogenous protein in the resulting transfected platelet cells.

Lipid nanoparticles of the present invention comprise a mRNA encoding an exogenous protein and a lipid mixture comprising an ionizable cationic lipid (ICL), a helper lipid, a sterol, and a polyethylene glycol (PEG)-lipid conjugate. The inventors have found novel lipid mixtures of specific ICLs and helper lipids which, when combined with a sterol and a PEG-lipid conjugate, enable the expression of exogenous proteins in transfected platelet cells. These specific ICLs and helper lipids are shown in Table 1:

The LNPs of the present invention may include one or more of the following ICLs in the lipid mixture: CL4H6, SM-102, ALC-0315, CL1H6, CL15H6, CL1D6, ALC-0159 or any combination thereof. For example, the lipid mixture may include CL4H6 or ALC-0315 as the ICL.

The lipid mixture in the LNPs of the present invention may include one or more of the following helper lipids: phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM), or any combination thereof. For example, the lipid mixture may include phosphatidylcholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as the helper lipid.

The LNPs of the present invention may include one or more of the following sterols in the lipid mixture: cholesterol or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, beta-sitosterol, fucosterol, or any mixture thereof. For example, the LNPs of the present invention may include cholesterol in the lipid mixture.

The LNPs of the present invention may include one or more of the following polyethylene glycol (PEG)-lipid conjugate in the lipid mixture: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE-PEG2000), or PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG). For example, the LNPs of the present invention may include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) in the lipid mixture.

Suitable lipid mixtures for use in the LNPs of the present invention may comprise, for example, CL4H6 as the ICL in combination with phosphatidylcholine (POPC) as the helper lipid. Other suitable lipid mixtures for use in the LNPs of the present invention may comprise, for example, ALC-0315 as the ICL and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as the helper lipid.

In the lipid mixture of the LNP of the present invention, the ICL, helper lipid, sterol and PEG-lipid conjugate are mixed to achieve a desired mole percent of each component. For example, the mole percent of the ICL may range from 30% to 55%, or any mole percent therebetween, for example the mole percent of the ICL may be 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, or any value therebetween; the mole percent of the helper lipid may range from 5% to 20%, or any mole percent therebetween, for example, the mole percent of the helper lipid may be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or any value therebetween; the mole percent of the sterol may range from 25% to 50%, or any mole percent therebetween, for example, the mole percent of the sterol may be 25%, 27.5%, 30%, 32.5%, 35%, 36%, 37%, 38%, 38.5%, 39%, 39.5%, 40%, 42%, 44%, 46%, 48%, 50%, or any value therebetween; and the mole percent of polyethylene glycol (PEG)-lipid conjugate may range from 0.5% to 3%, or any mole percent therebetween, for example, the mole percent of polyethylene glycol (PEG)-lipid conjugate may be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or any value therebetween. For example, the mole percent of the ICL be 50%; the mole percent of the helper lipid may be 10%; the mole percent of the sterol may be 38.5%; and the mole percent of polyethylene glycol (PEG)-lipid conjugate may be 1.5%.

In some embodiments, the lipid mixture in the LNP particles comprises 50 mole percent CL4H6, 10 mol % phosphatidylcholine (POPC), 38.5% cholesterol, and 1.5% 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). In other embodiments, the lipid mixture in the LNP particles comprises 50 mole percent ALC-0315, 10 mol % 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 38.5% cholesterol, and 1.5% 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000.

The LNPs of the present invention, in addition to the above-described lipid mixture, also comprise messenger RNA (mRNA). The messenger RNA may encode an exogenous protein that is not inherent to or produced by unmodified platelet cells. Examples of exogenous proteins that may be encoded by the mRNA contained within the LNP include, but are not limited to: a coagulation factor, an antifibrinolytic protein, an anticoagulant, a fibrinolytic protein, an antimicrobial protein, an immunomodulator, an anti-cancer protein, a genetic editing protein, and a reporter protein. Alternatively, the messenger RNA may not encode an exogenous protein but may be an RNA or DNA aptamer molecule designed or selected to bind to, or otherwise effect the activity of, a specific target biomolecule.

Lipid nanoparticles (LNP) are a flexible and clinically approved platform which enable delivery and intracellular release of genetic material and several methods have been developed in recent years (Novakowski et al, 2019, Scientific Reports 9:552; Sato et al., 2019, J. Controlled Release, 295 (10): 140-152).